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Libera Lo Presti, Bernd Zechmann, Jochen Kumlehn, Liang Liang, Daniel Lanver, Shigeyuki Tanaka, Ralph Bock, Regine Kahmann
Successful colonization of plants by prokaryotic and eukaryotic pathogens requires active effector-mediated suppression of defense responses and host tissue reprogramming. Secreted effector proteins can either display their activity in the apoplast or translocate into host cells and function therein. Although characterized in bacteria, the molecular mechanisms of effector delivery by fungal phytopathogens remain elusive. Here we report the establishment of an assay that is based on biotinylation of effectors in the host cytoplasm as hallmark of uptake...
September 26, 2016: New Phytologist
Elisabeth Schmidtmann, Tobias Anton, Pascaline Rombaut, Franz Herzog, Heinrich Leonhardt
Chromatin structure and function are determined by a plethora of proteins whose genome-wide distribution is typically assessed by immunoprecipitation (ChIP). Here, we developed a novel tool to investigate the local chromatin environment at specific DNA sequences. We combined the programmable DNA binding of dCas9 with the promiscuous biotin ligase BirA* (CasID) to biotinylate proteins in the direct vicinity of specific loci. Subsequent streptavidin-mediated precipitation and mass spectrometry identified both known and previously unknown chromatin factors associated with repetitive telomeric, major satellite and minor satellite DNA...
September 27, 2016: Nucleus
Ellen Margrethe Haugsten, Vigdis Sørensen, Michaela Kunova Bosakova, Gustavo Antonio de Souza, Pavel Krejci, Antoni Wiedlocha, Jørgen Wesche
The fibroblast growth factor receptors (FGFRs) are important oncogenes promoting tumor progression in many types of cancer, such as breast, bladder, and lung cancer as well as multiple myeloma and rhabdomyosarcoma. However, little is known about how these receptors are internalized and down-regulated in cells. We have here applied proximity biotin labeling to identify proteins involved in FGFR4 signaling and trafficking. For this purpose we fused a mutated biotin ligase, BirA*, to the C-terminal tail of FGFR4 (FGFR4-BirA*) and the fusion protein was stably expressed in U2OS cells...
October 7, 2016: Journal of Proteome Research
Selma Waaijers, Javier Muñoz, Christian Berends, João J Ramalho, Soenita S Goerdayal, Teck Y Low, Adja D Zoumaro-Djayoon, Michael Hoffmann, Thijs Koorman, Roderick P Tas, Martin Harterink, Stefanie Seelk, Jana Kerver, Casper C Hoogenraad, Olaf Bossinger, Baris Tursun, Sander van den Heuvel, Albert J R Heck, Mike Boxem
BACKGROUND: Affinity purification followed by mass spectrometry (AP/MS) is a widely used approach to identify protein interactions and complexes. In multicellular organisms, the accurate identification of protein complexes by AP/MS is complicated by the potential heterogeneity of complexes in different tissues. Here, we present an in vivo biotinylation-based approach for the tissue-specific purification of protein complexes from Caenorhabditis elegans. Tissue-specific biotinylation is achieved by the expression in select tissues of the bacterial biotin ligase BirA, which biotinylates proteins tagged with the Avi peptide...
2016: BMC Biology
Santhosh M Nadipuram, Elliot W Kim, Ajay A Vashisht, Andrew H Lin, Hannah N Bell, Isabelle Coppens, James A Wohlschlegel, Peter J Bradley
UNLABELLED: Toxoplasma gondii is an obligate intracellular parasite that invades host cells and replicates within a unique parasitophorous vacuole. To maintain this intracellular niche, the parasite secretes an array of dense granule proteins (GRAs) into the nascent parasitophorous vacuole. These GRAs are believed to play key roles in vacuolar remodeling, nutrient uptake, and immune evasion while the parasite is replicating within the host cell. Despite the central role of GRAs in the Toxoplasma life cycle, only a subset of these proteins have been identified, and many of their roles have not been fully elucidated...
2016: MBio
Sarah K Henke, John E Cronan
Group II biotin protein ligases (BPLs) are characterized by the presence of an N-terminal DNA binding domain that functions in transcriptional regulation of the genes of biotin biosynthesis and transport. The Staphylococcus aureus Group II BPL which is called BirA has been reported to bind an imperfect inverted repeat located upstream of the biotin synthesis operon. DNA binding by other Group II BPLs requires dimerization of the protein which is triggered by synthesis of biotinoyl-AMP (biotinoyl-adenylate), the intermediate in the ligation of biotin to its cognate target proteins...
November 2016: Molecular Microbiology
Fengchun Zhao, Yuan Tian, Huimin Wang, Jiye Liu, Xiao Han, Zhengyou Yang
Organophosphorus pesticides (OPs) are the most widely used pesticides in agriculture, and OP residues have been broadly reported in food and environmental samples. The aim of this study is to develop a recombinant antibody-based broad-specificity immunoassay for OPs. A phage display library was prepared from a mouse pre-immunized with a generic immunogen of OPs, and a single-chain variable fragment (scFv) antibody was selected. The selected scFv antibody was fused with biotin acceptor domain (BAD) and overexpressed as an inclusion body in Escherichia coli BL21 (DE3)...
September 2016: Analytical and Bioanalytical Chemistry
Renata Varnaitė, Stuart A MacNeill
Proximity-dependent biotin identification (BioID) is a recently developed method that allows the identification of proteins in the close vicinity of a protein of interest in living cells. BioID relies on fusion of the protein of interest with a mutant form of the biotin ligase enzyme BirA (BirA*) that is capable of promiscuously biotinylating proximal proteins irrespective of whether these interact directly or indirectly with the fusion protein or are merely located in the same sub-cellular neighbourhood. The covalent addition of biotin allows the labelled proteins to be purified from cell extracts on the basis of their affinity for streptavidin and identified by mass spectrometry...
June 22, 2016: Proteomics
Huiyan Ye, Mingzhu Cai, Huimin Zhang, Zhencui Li, Ronghui Wen, Youjun Feng
Biotin protein ligase is universal in three domains of life. The paradigm version of BPL is the Escherichia coli BirA that is also a repressor for the biotin biosynthesis pathway. Streptococcus suis, a leading bacterial agent for swine diseases, seems to be an increasingly-important opportunistic human pathogen. Unlike the scenario in E. coli, S. suis lacks the de novo biotin biosynthesis pathway. In contrast, it retains a bioY, a biotin transporter-encoding gene, indicating an alternative survival strategy for S...
2016: Scientific Reports
Guanghui Li, Min Zhu, Lu Ma, Junrong Yan, Xiaoling Lu, Yanfei Shen, Yakun Wan
A phage display library of variable domain of the heavy chain only antibody or nanobody (Nb) was constructed after immunizing a bactrian camel with testosterone. With the smaller molecular size (15 kDa), improved solubility, good stability, high affinity, specificity, and lower immunogenicity, Nbs are a promising tool in the next generation of diagnosis and medical applications. Testosterone is a reproductive hormone, playing an important role in normal cardiac function and being the highly predictive marker for many diseases...
June 8, 2016: ACS Applied Materials & Interfaces
Huimin Wang, Fengchun Zhao, Xiao Han, Zhengyou Yang
In this article, we reported the development of a biotinylated single-chain variable fragment (scFv) antibody based indirect competitive enzyme-linked immunosorbent assay (IC-ELISA) for parathion-methyl (PM) detection. Firstly, a phage display library was generated using a pre-immunized BALB/C mouse against a specific hapten of PM. After four rounds of panning, the scFv gene fragments were transferred into a secreted expression vector. Then, the scFv antibodies were secreted expressed and screened by IC-ELISA against PM...
October 2016: Protein Expression and Purification
I N Sheremetyeva, I V Kartavtseva, L V Frisman, T V Vasil'eva, A V Adnagulova
The genetic variability of the mitochondrial DNA control region sequences was estimated for the Maximowicz's vole Microtus maximowiczii from the Middle Amur River region located between the confluence of Amur River with Ussuri River and Zeya River. The species as a whole was characterized by a high level of genetic variability. For each individual sample, low nucleotide diversity was observed, except for two samples in which a more than twofold increase in this index was revealed. The presence of the contact zone of two genetically distinct populations in the area between Bira and Bidzhan rivers is suggested...
October 2015: Genetika
Huimin Zhang, Qingjing Wang, Derek J Fisher, Mingzhu Cai, Vandana Chakravartty, Huiyan Ye, Ping Li, Jose O Solbiati, Youjun Feng
Biotin protein ligase (BPL) is widespread in the three domains of the life. The paradigm BPL is the Escherichia coli BirA protein, which also functions as a repressor for the biotin biosynthesis pathway. Here we report that Lactococcus lactis possesses two different orthologues of birA (birA1_LL and birA2_LL). Unlike the scenario in E. coli, L. lactis appears to be auxotrophic for biotin in that it lacks a full biotin biosynthesis pathway. In contrast, it retains two biotin transporter-encoding genes (bioY1_LL and bioY2_LL), suggesting the use of a scavenging strategy to obtain biotin from the environment...
2016: Scientific Reports
Lydia N Horn, Habteab M Ghebrehiwot, Hussein A Shimelis
Cowpea (Vigna unguiculata [L.] Walp.) yields are considerably low in Namibia due to lack of improved varieties and biotic and abiotic stresses, notably, recurrent drought. Thus, genetic improvement in cowpea aims to develop cultivars with improved grain yield and tolerance to abiotic and biotic stress factors. The objective of this study was to identify agronomically desirable cowpea genotypes after mutagenesis using gamma irradiation. Seeds of three traditional cowpea varieties widely grown in Namibia including Nakare (IT81D-985), Shindimba (IT89KD-245-1), and Bira (IT87D-453-2) were gamma irradiated with varied doses and desirable mutants were selected from M2 through M6 generations...
2016: Frontiers in Plant Science
Svetlana P Ikonomova, Ziming He, Amy J Karlsson
Antibody fragments, such as the single-chain variable fragment (scFv), have much potential in research and diagnostics because of their antigen-binding ability similar to a full-sized antibody and their ease of production in microorganisms. Some applications of antibody fragments require immobilization on a surface, and we have established a simple immobilization method that is based on the biotin-streptavidin interaction and does not require a separate purification step. We genetically fused two biotinylation tags-the biotin carboxyl carrier protein (BCCP) or the AviTag minimal sequence-to six different scFvs (scFv13R4, scFvD10, scFv26-10, scFv3, scFv5, and scFv12) for site-specific biotinylation in vivo by endogenous biotin ligases produced by Escherichia coli...
August 2016: Journal of Immunological Methods
Christoph Schweingruber, Paolo Soffientini, Marc-David Ruepp, Angela Bachi, Oliver Mühlemann
Proximity-dependent trans-biotinylation by the Escherichia coli biotin ligase BirA mutant R118G (BirA*) allows stringent streptavidin affinity purification of proximal proteins. This so-called BioID method provides an alternative to the widely used co-immunoprecipitation (co-IP) to identify protein-protein interactions. Here, we used BioID, on its own and combined with co-IP, to identify proteins involved in nonsense-mediated mRNA decay (NMD), a post-transcriptional mRNA turnover pathway that targets mRNAs that fail to terminate translation properly...
2016: PloS One
Anastassia K Pogoutse, Christine Chieh-Lin Lai, Nicholas Ostan, Rong-hua Yu, Anthony B Schryvers, Trevor F Moraes
Obtaining accurate kinetics and steady-state binding constants for biomolecular interactions normally requires pure and homogeneous protein preparations. Furthermore, in many cases, one of the ligands must be labeled. Over the past decade, several technologies have been introduced that allow for the measurement of kinetics constants for multiple different interactions in parallel. One such technology is bio-layer interferometry (BLI), which has been used to develop systems that can measure up to 96 biomolecular interactions simultaneously...
May 15, 2016: Analytical Biochemistry
Akhee S Jahan, Maxime Lestra, Lee Kim Swee, Ying Fan, Mart M Lamers, Fikadu G Tafesse, Christopher S Theile, Eric Spooner, Roberto Bruzzone, Hidde L Ploegh, Sumana Sanyal
Posttranslational modifications are central to the spatial and temporal regulation of protein function. Among others, phosphorylation and ubiquitylation are known to regulate proximal T-cell receptor (TCR) signaling. Here we used a systematic and unbiased approach to uncover deubiquitylating enzymes (DUBs) that participate during TCR signaling in primary mouse T lymphocytes. Using a C-terminally modified vinyl methyl ester variant of ubiquitin (HA-Ub-VME), we captured DUBs that are differentially recruited to the cytosol on TCR activation...
February 9, 2016: Proceedings of the National Academy of Sciences of the United States of America
Kaitlynn R Davenport, Christopher A Smith, Heike Hofstetter, James R Horn, Oliver Hofstetter
In this study, the effect of random vs. site-directed immobilization techniques on the performance of antibody-based HPLC columns was investigated using a single-domain camelid antibody (VHH) directed against methotrexate (MTX) as a model system. First, the high flow-through support material POROS-OH was activated with disuccinimidyl carbonate (DSC), and the VHH was bound in a random manner via amines located on the protein's surface. The resulting column was characterized by Frontal Affinity Chromatography (FAC)...
May 15, 2016: Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences
Petros Batsios, Irene Meyer, Ralph Gräf
The identification of a bona fide lamin-like protein in Dictyostelium made this lower eukaryote an attractive model organism to study evolutionarily conserved nuclear envelope (NE) proteins important for nuclear organization and human laminopathies. Proximity-dependent biotin identification (BioID), reported by Roux and colleagues, is a powerful discovery tool for lamin-associated proteins. In this method, living cells express a bait protein (e.g., lamin) fused to an R118G-mutated version of BirA, an Escherichia coli biotinylase...
2016: Methods in Enzymology
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